Differential activation of endogenous TGF-β and BMP signaling between FOB and POb cells.

(A) Immunoblotting analysis using specific anti-phosphoSmad-2 and phoshoSmad-1/5 antibodies shows a more intense phosphorylation of Smad-2 in POb than FOb cells. In contrast, analysis with anti-phoshoSmad-1/5 antibody reveals a stronger staining in FOb. To assess for the total amount of endogenous Smad-2 and Smad-1/5 and to control for equal loading and transfer of the samples membranes were reprobed with anti-Smad-2, Smad-1/5 and anti-α-Tubulin antibodies. Histogram below represents quantification of phosphorylated Smad-2 and Smad-1/5 proteins obtained by Image J program. The relative intensity of each band was normalized to their respective α-Tubulin loading controls. (B) immunofluorescent staining using anti-phosphoSmads antibodies as above confirms the results obtained by immunoblotting analysis. Immunofluorescent staining using anti-phosphoBcl-2 antibody detects higher levels of the anti-apoptotic protein Bcl-2 in FOb compared to POb. Dapi nuclear counterstaining. (C) Immunoblotting analysis performed as above (A) showing that the differential activation of the two signaling pathways observed between FOb and POb cells is maintained throughout their osteogenic differentiation.